Interleukin-35 promotes Breg expansion and interleukin-10 production in CD19(+) B cells in patients with ankylosing spondylitis

OBJECTIVE: IL-35 is a potent immunosuppressive and anti-inflammatory cytokine, consisting of a p35 subunit and an Epstein–Barr virus–induced gene 3 (EBI3) subunit, which suppresses CD4(+) effector T cell proliferation and promotes regulatory T cell (Treg) expansion. However, the effects of IL-35 on regulatory B cells (Bregs) in ankylosing spondylitis (AS) have not been explored. The present study aimed (i) to measure serum IL-35 levels and the percentages of Bregs in the peripheral blood of patients with AS and (ii) to explore their relationships in the pathogenesis of AS. METHODS: A total of 77 patients with AS (AS group), including 47 inactive AS and 30 active AS cases, and 59 healthy controls (HCs) were enrolled into this study. The serum levels of IL-35 and IL-10 were detected by ELISA, and the mRNA levels of p35 and EBI3 were measured by RT–qPCR. The percentages of CD19(+)CD24(hi)CD38(hi) and CD19(+)CD24(hi)CD27(+) Bregs and IL-35 receptor (IL-12Rβ2, IL-27Rα and gp130), IL-10, p-STAT1, p-STAT3, and p-STAT4 in CD19(+) B cells were detected by flow cytometry. The correlations between IL-35 levels and percentages of Bregs were analyzed by determining Pearson’s correlation coefficient. The effect of IL-35 on Bregs was determined by mix-culture of recombinant (r) IL-35 with peripheral blood mononuclear cells (PBMCs). RESULTS: The serum IL-35 and IL-10 levels, p35 and EBI3 mRNA levels, and the percentages of CD19(+)CD24(hi)CD38(hi) and CD19(+)CD24(hi)CD27(+) Bregs were significantly lower in AS patients than those in HCs. In addition, the percentages of CD19(+)CD24(hi)CD38(hi) and CD19(+)CD24(hi)CD27(+) Bregs in active AS patients were significantly lower than those in inactive AS patients. The serum IL-35 levels were positively correlated with the percentages of CD19(+)CD24(hi)CD38(hi) and CD19(+)CD24(hi)CD27(+) Bregs in AS patients. IL-12Rβ2 and IL-27Rα, but not gp130 subunit, were expressed in CD19(+) B cells in AS patients. RIL-35 could effectively promote CD19(+)CD24(hi)CD38(hi) Breg expansion and IL-10 production. Meanwhile, rIL-35 also promoted the expression of IL-12Rβ2 and IL-27Rα and the phosphorylation of STAT1 and STAT3 in CD19(+) B cells. CONCLUSION: These results demonstrated that reduced IL-35 production may be associated with Bregs defects in AS patients. RIL-35 induced the proliferation of CD19(+)CD24(hi)CD38(hi) Bregs and IL-10 production, suggesting that IL-35 may serve as a reference for further investigation to develop novel treatments for AS.